Abstract 3309: Self-assembling and disassembling (SADA) domain is critical to the binding, and anti-tumor efficacy of GD2-SADA
Authors: Brian H. Santich, Stine Louise Kjellev, Sarah Cheal, Linlin Wang, Mette Gillberg, Mallika Vadlamudi, Amadou A. Ouattara, Nico Liebenberg, Lone Frost Larsen, Darren R. Veach, Shin Seo, Nai-Kong V. Cheung, Steven M. Larson, Steen Lisby
Journal: Cancer Research (2022)

Abstract Introduction: GD2-SADA is a pre-targeted radioimmunotherapy drug-candidate designed to target radioactive lutetium-177 (in the form of 177Lu-DOTA) to GD2-expressing tumor cells (GD2-SADA:177Lu-DOTA Drug Complex). GD2-SADA is comprised of an anti-GD2 single-chain variable fragment (scFv), an anti-DOTA scFv that binds specifically to DOTA metal chelates, such as 177Lu-DOTA, and a SADA domain based on the human P53 tetramerization sequence, which can self-assemble from a monomeric 60-kDa polypeptide into 240-kDa tetrameric protein. We have previously demonstrated (1) that GD2-SADA has a unique clearance profile that allowed for the delivery of 177Lu-DOTA to tumors with minimal exposure to normal tissues like the bone marrow or kidneys and shrank established neuroblastoma in preclinical mouse models. We now provide additional mechanistic data demonstrating the importance of the SADA domain in providing this potent anti-tumor function. Methods: To study the role of the SADA domain, we designed a modified version of GD2-SADA that could not self-assemble into a tetrameric state by removing the entire SADA domain. The resulting P53(-/-)GD2-SADA protein retained binding to GD2 and Lu-DOTA but remained in a 60 kDa monomeric state in assays. P53(-/-)GD2-SADA and GD2-SADA were compared using SPR to measure antigen binding affinities, flow cytometry to evaluate cell binding, and in preclinical mouse models to evaluate tumor uptake and anti-tumor efficacy. Results: GD2-SADA demonstrated stronger binding affinity to GD2 antigen compared to P53(-/-)GD2-SADA, but comparable binding affinity to Lu-DOTA. In SPECT/CT imaging studies using tumor bearing mice, GD2-SADA demonstrated substantially higher uptake and persistence in GD2-expressing tumors compared with P53(-/-)GD2-SADA. Finally, treatment of tumor bearing mice with 3 cycles of GD2-SADA or P53(-/-)GD2-SADA and 177Lu-DOTA (once per week each for 3 weeks) resulted in potent anti-tumor responses from GD2-SADA, with only modest anti-tumor efficacy from P53(-/-)GD2-SADA at two different dose levels corresponding to equal molar doses (1 GD2-SADA to 1 P53(-/-)GD2-SADA) or equal mass doses (1 GD2-SADA to 4 P53(-/-)GD2-SADA i.e. same number of binding sites). Conclusions: The tetramerizing function of the SADA domain is critically important to the binding activity and anti-tumor efficacy of GD2 SADA. These data confirm that the SADA domain increases tumor antigen binding, uptake and persistence in tumor tissue, and markedly improves anti-tumor responses in preclinical models. References: Santich BH, Cheal SM, Ahmed M, McDevitt MR, Ouerfelli O, Yang G, et al. A Self-Assembling and Disassembling (SADA) Bispecific Antibody (BsAb) Platform for Curative Two-step Pretargeted Radioimmunotherapy. Clin Cancer Res Off J Am Assoc Cancer Res. 2021 Jan 15;27(2):532-41. Citation Format: Brian H. Santich, Stine Louise Kjellev, Sarah Cheal, Linlin Wang, Mette Gillberg, Mallika Vadlamudi, Amadou A. Ouattara, Nico Liebenberg, Lone Frost Larsen, Darren R. Veach, Shin Seo, Nai-Kong V. Cheung, Steven M. Larson, Steen Lisby. Self-assembling and disassembling (SADA) domain is critical to the binding, and anti-tumor efficacy of GD2-SADA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3309.